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June 18, 2019

University of Bristol

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Abstract

There are greater than 500 protein kinases, many of which have been shown to be dysregulated in disease states and comprise greater than 30% of all drug development. Harnessing chelation-enhanced fluorescence (CHEF) with the sulfonamido-oxine (Sox) chromophore in peptide or protein substrates has created a simple yet powerful method to measure the activity of protein kinases using a direct, homogeneous and continuous (kinetic) format. This kinetic assay platform allows elucidation of drug mechanism of action and is increasingly being applied earlier in the drug development process to address challenges and opportunities for next generation kinase inhibitors.

Introduction

Protein kinases and phosphatases are ubiquitous in nature and are critical enzymes that regulate the complexity of signal transduction pathways in normal and disease states. A major focus of all research efforts is addressing drug development for these enzymes that spans many disease areas1-3. Kinases form a class of signaling enzymes that carry out the transfer of a phosphate group from ATP to a serine, threonine or tyrosine residue with the target protein, which can dramatically change the activity, location within a cell, and the nature of the signaling complexes formed with that protein. The protein phosphatases catalyze the removal of the phosphate from the target protein, thereby creating a dynamic balance. Both enzyme classes regulate diverse cellular functions including cell growth, differentiation, metabolism, proliferation and survival. Abnormal signaling in any one of these pathways can have severe consequences resulting in a diseased state.

The ability to accurately and precisely quantify kinase activity and define the effects of compounds that modulate this activity is essential for understanding the complex biology of phosphoregulation and for the development and monitoring of effective therapeutic agents against these enzymes4-8. Precise activity measurements are also critical for understanding the effects of mutations on enzyme function in determining signal transduction and cellular fate decisions9,10.

A kinetic kinase assay platform has been developed by AssayQuant Technologies, Inc. that provides a simple, one-step homogeneous, fluorescence-based assay for rapid, sensitive and continuous detection of serine/threonine and tyrosine kinase activities. The assay directly measures the catalytic activity of target kinases using optimized peptide substrates under optimal conditions including pH, selected metal ion cofactors, and low to physiological (mM) ATP concentrations allowing both ATP-competitive and ATP non-competitive (allosteric) kinase inhibitors to be selected and characterized. Here we demonstrate utility across a variety of applications. Although this article focuses on measuring protein kinase activity, the platform also allows study of protein phosphatases using the appropriate phosphopeptide substrates11,12.

Assay Principle

The Chelation-Enhanced Fluorescence (ChEF) method for protein kinase sensing was invented by the Imperiali laboratory at the Massachusetts Institute of Technology (MIT) and has been extensively validated13-16. The ChEF sensing mechanism exploits a synthetic α-amino acid with a side chain bearing an 8-hydroxyquinoline derivative (sulfonamido-oxine, Sox), which upon coordination to Mg(II), relays information on the phosphorylation state of proximal serine,threonine or tyrosine residues in peptide- and proteinbased kinase substrates (Figure 1). In the absence of phosphorylation, Sox shows low affinity for Mg(II); upon phosphorylation, Mg(II) affinity is enhanced due to the advantageous chelate effect, involving the Sox and the introduced phosphate group, and fluorescence is turned on. Since it was first introduced, the Imperiali laboratory has continually improved the technology for the ChEF method13-21. AssayQuant Technologies, Inc., who holds the exclusive license for the technology, has been systematically integrating improvements from MIT and through in house innovation to deliver optimum performance of their PhosphoSens® product line, which currently includes 167 validated protein kinases and 27 Sox-based substrates (www.assayquant. com). Assays can be performed in continuous kinetic mode, using commonly available fluorescence plate readers in 96-, 384- and 1536-well plate formats and a wide range of sample types including recombinant enzymes, immunoprecipitated kinases, and crude cell or tissue lysates13-23. The platform is ideal for understanding mechanism of action, but is increasingly being applied earlier in the drug development process to define and optimize next generation kinase inhibitors by enabling precise determinations of kinase activity regulation, kinase inhibitor screening, structure-activity relationships (SAR), and inhibitor parameter determinations (IC50, Ki, kinact, residence time, mechanism of action)24-27.

Figure 2A illustrates typical fluorescence changes upon phosphorylation of a peptide substrate and Figure 2B shows excitation and emission spectra of a typical phosphorylated peptide. The ƛExMax of the chelated Mg(II) with the 8-hydroxyquinoline is 360 nm and the ƛEmMax is 485 nm. Since the fluorescence emission spectrum is relatively broad (see Figure 2B), fluorescence emission can be monitored between 475-505 nm with minimal loss of signal intensity. Efforts to extend the emission to >600 nm are in progress.

Figure 2. Fluorescence Spectra of PhosphoSens® Peptides. (A) Typical fluorescence changes upon phosphorylation of a peptide substrate for ERK1/2 MAPK: Ac-VPLL-pT-PG-[CSox]-RRG-COOH. (B) Excitation and Emission spectra. The ƛExMax of chelated Mg(II) with the 8-hydroxyquinoline is 360 nm and the ƛEmMax is 485 nm.

Materials and Methods

Materials

Materials were sourced and prepared as per the manufacturers’ recommendations. All kinases were from Carna Biosciences (www.carnabio.com). Briefly, the assay reaction was initiated by the addition of a master mix containing either a protein kinase or the CSox-peptide substrate to a well containing the final component needed to start the reaction. Typical final concentrations of each reaction component are as follows: 50 mM HEPES, pH 7.5, 1 mM ATP (or adjusted as needed), 1 mM DTT, 0.01% Brij-35, 0.5 mM EGTA, 10 mM MgCl2, 10 μM peptide substrate, 0.1-10 nM kinase (or adjusted as needed), Additional co-factors or additives (as needed).

The peptide substrate was resuspended to create a 1 mM (100x) stock. For both peptide and ATP, a 10x working solution was prepared as necessary in ultrapure H2O just before use. DTT working solution, 10x, was prepared from 1000x stock in ultrapure H2O just before use. Kinase reaction master mix was prepared as per manufacturers’ instructions. Kinases were prepared as a 5x working concentration just before use in kinase dilution buffer and added to the wells to initiate reactions. Fluorescent measurements were taken at 30 ºC either kinetically or as an end point measurement.

Kinase titrations were prepared as an 11-point, 2-fold serial dilution using 1x kinase dilution buffer as well as a no-kinase control. Each dilution in the series represented 5x the final concentration of kinase in the reaction resulting in a concentration range that spans 3 log units (e.g., from 20 nM to 20 pM). Samples and standards were plated in triplicate. All assays were performed in Corning half-area, 96-well, white NBS plates (P/N 3824).

Instrumentation

A Synergy™ Neo2 (BioTek Instruments, Inc., Winooski, VT) was used for reading fluorescence intensity in kinetic mode with an excitation wavelength (ƛex) of 360 nm and an emission wavelength (ƛem) of 485 nm. Readings were typically performed every 30 seconds for 60 minutes or every 2-3 minutes for 150 minutes.

Data reduction

The background fluorescence was subtracted for each time point from the total fluorescence signal to obtain corrected relative fluorescence units (RFU) values. The corrected RFUs vs. time was plotted. The slope of the initial linear portion of each curve was determined, which is the initial reaction rate (RFU/ min., or converted to RFU/pmole kinase/min.). Reaction rates from linear or non-linear (kinases that exhibit a lag phase) fit of the data were generated using Gen5™ software (BioTek Instruments, Inc., Winooski, VT) or by exporting data to GraphPad Prism (La Jolla, CA).

Background: Kinase Kinetic Parameter Analysis

To determine Km, a fixed concentration of kinase was combined with a serial dilution of a CSox-based peptide substrate. Km determinations require a range of substrate concentrations, ideally from 0.1- to 2 to 10-fold of the estimated Km. Vmax values in μmol.mg-1min-1 were determined as described below and presented in detail in Lukovic et al.16.

To determine Vmax, from the initial rates of product formation, a correction for the decrease in fluorescence intensity due to the substrate consumption was required. The fluorescence intensity at any given time in the reaction is determined from the following equation:

(1)

where I(t) is the fluorescence intensity, S(t) is the amount of substrate in μM, P(t) is the amount of product in μM, ƒs is the fluorescence intensity per μM of substrate, and ƒp is fluorescence intensity per μM of product.

The amount of substrate and product at any given point are related by:

(2)

where S0 is the initial amount of substrate.

Substitution of eq. (2) into eq. (1) followed by rearrangement yields:

(3)

The initial velocity of the reaction is the change in the amount of product over time, so taking the derivative of eq. (3) with respect to time gives:

(4)

The initial slope of the reaction, dI(t)/dt, should be measured within the first 10% of substrate turnover to ensure initial rate analysis. The constants ƒP and ƒS were calculated from the standard curves of RFU versus concentration of P and S, respectively. These values will depend on the concentration of Mg2+ and the Mg2+ dissociation constant of each peptide and were determined empirically under the desired assay conditions. The Km and Vmax were determined from a direct, non-linear fit of ν vs. [S] plots using the Michaelis-Menten equation:

(5)

These determinations are routinely performed using Graphpad Prism Software.

The phosphopeptide standard curve based on enzymatic synthesis of the product was generated by combining a fixed concentration of kinase with a serial dilution of CSox peptide substrate at the same concentrations used in the kinetic assay. Typically, the amount of kinase used to generate the phosphopeptide standard curve is higher than the concentration used in the kinetic analysis in order to ensure complete phosphorylation of the peptide substrate. The kinase concentration required to reach signal saturation will vary between kinases. A titration of kinase was performed, as previously described, with the highest concentration of peptide substrate to determine the amount of kinase required to achieve complete phosphorylation.

Results

CSox-based Substrate Optimization for EGFR RTK

The Epidermal Growth Factor Receptor (EGFR) is a Receptor Tyrosine Kinase (RTK) that has been intensely studied to understand its complex biology, the generation of kinase inhibitors, the appearance of mutations that confer drug resistance and to define the best sequence of treatments for human cancer24,26-29. To develop improved Sox-based assays for the EGFR, partially degenerate peptide libraries, incorporating the Sox chromophore, were used to screen for sensors with increased reaction rates. Reactions conditions were optimized and two rounds of selection were performed.

Reactions were incubated at 30 °C for 120 minutes and fluorescent intensity data were collected every 5 minutes and plotted as relative fluorescent units corrected for background (RFU, corrected) versus time (Figure 3). Background subtraction was achieved by subtracting RFU values from a no kinase control at each time point with the same peptide concentration from each kinase reaction. Corrected RFU values were plotted versus time for determination of the initial reaction velocities (slope of the line; RFU/min.) from the linear portion of each curve. Each line in the graph represents a different sequence consisting of either a single amino acid substitution (Round 1) or combinations of favorable substitutions (Round 2) as identified in Round 1.

Figure 3. Optimization of degenerate peptide sequences. Two rounds of selection were performed with partially degenerate Sox-peptide libraries. The steeper the curve, the higher the reaction rate.

This approach allowed the selection of optimum Sox-based sequences for measurement of wild-type EGFR and each of the clinically-relevant mutants (Figure 4). These data demonstrate the dramatic increase in kinase activity observed with each of these clinically-relevant mutants, which drives tumor growth and metastasis and resistance to first-line drugs.

Figure 4. Progress curves generated using the PhosphoSens® AQT0099 Sox-based substrate incubated with 5 nM of the wild-type EGFR and each of the clinical variants.

Enzyme Titration

Kinase titration reactions were performed with selected peptide substrates to determine linearity. Reactions were incubated at 30 °C for 150 minutes and fluorescent intensity data were collected every 3 minutes and plotted versus time. Each line represents a different enzyme concentration (Figure 5a and 5c). Initial velocity determinations were calculated for each reaction and plotted versus each enzyme concentration. The data demonstrate linearity across a range of kinase concentrations from 10 nM down to 160 pM for the wild-type EGFR and from 1.3 nM down to 39 pM for the EGFR (T790M/L858R) (Figure 5b and 5d), highlighting the dramatic increase in activity of this mutant variant. Any kinase concentration that provided a linear signal with time and resulted in <10% of the substrate being phosphorylated were used 4 for subsequent experiments.

Assay performance was examined to determine the impact of varying the final reaction volume (25, 50 and 75 μL) and EGFR concentration (0.25 to 1.25 nM) on precision determined by calculation of Z’ values (Figure 6, Table 1). A final reaction volume of 50 μL is recommended by the manufacturer, however, the final choice is based on assay requirements (i.e., enzyme and substrate concentration, nature of inhibitor studies, etc.).

Figure 6. Effect of assay volume on precision with Z’ values plotted against EGFR concentration for each assay volume.

Table 1. Z' Values for Different Final Reaction Volumes and EGFR Concentrations. Bolded Z’ values correspond to the Lower Limit of Quantitation (LLOQ), which is the lowest concentration of enzyme where the CV is still <20%. Assay formats with Z' values >0.5 are considered to be robust. The PhosphoSens® assay format typically delivers Z' values >0.7, even at very low enzyme concentrations, highlighting the very high precision of the Sox-based assay platform.

Substrate Titration for Km and Vmax Determination

The reaction velocities for each substrate concentration were then determined by incubating a 1.5-fold serial dilution of peptide substrate, 200 to 2.3 μM, with a constant concentration of each EGFR. Each dilution series was analyzed in triplicate as described above (Figures 7 and 8).

Generation of a phosphopeptide standard curve was performed as described for Figure 8, but using 20- fold higher levels of the EGFR (T790M/L858R) in the reaction to ensure that all of the PhosphoSens® Peptide Substrate AQT0099 was converted to its phosphorylated form (Figure 9). The standard curves were constructed by plotting the saturating RFU values for each sample versus the concentration of the peptide substrate in the reaction (Figure 10). The slope of the standard curve was determined and used to convert reaction rate velocities. Velocities were converted to μM/min by dividing the reaction velocities (RFU/min) by the slope of the phosphopeptide standard curve (RFU/ μM) (Table 2). The converted velocities were plotted versus the substrate concentration and the data fit to the Michaelis-Menten equation for determination of Km and Vmax (Figure 11a and 11b). For EGFR the Km and Vmax were determined to be 79 ±1.9 μM and 0.32 ±0.0037 μM/min, respectively. For the EGFR (T790M/ L858R), the Km and Vmax were determined to be 92 ±5.0 μM and Vmax equal to 0.55 ±0.017 μM/min, respectively. Since the amount of substrate being used (10 μM) is much less than the Km, the difference in Km are less significant, however the fact that the Vmax for the EGFR (T790M/L858R) mutant under these conditions is 1.7-fold greater is dramatic and contributes to the cancer phenotype.

Figure 9. Phosphopeptide standard curve: 20 nM EGFR (T790M/ L858R) was used to completely phosphorylate all of the PhosphoSens® Peptide Substrate AQT0099 at each concentration to determine the saturating RFU values for each sample and to then generate a phosphopeptide standard curve.

Figure 10. The slope for this curve was determined to be 621 ±11 RFU Corrected/μM, which is used to convert reaction velocities from RFU/min to μM/min.

The resulting reaction rates (RFU/min.) values were normalized as a percentage of the untreated (no inhibitor) controls and plotted versus the Tarceva concentration (Figure 13) and the IC50 values were determined using a 4-parameter logistic curve fit. Note that each data point is the mean ±std dev. and the error bars aren't visible due to the high precision of the PhosphoSens® assay format.

For the EGFR, the IC50 value was determined to be 20 ±0.29 nM, which is similar to the IC50 value of 40 nM reported by Kitagawa, et al. (2012)30, determined under similar conditions with 1 mM ATP. The IC50 value determined for the EGFR (T790M/L858R) was 7.6 ±0.19 μM or 380-fold less sensitive to Tarceva, illustrating the resistance to drug that develops in patients.

Conclusion

The enabling features and benefits of the PhosphoSens® detection technology and the Synergy™ Neo2 HTS Multi-Mode Microplate Reader from Biotek Instruments, delivers a powerful combination with improved performance for drug discovery efforts for next generation protein kinase inhibitors. The platform delivers continuous (kinetic) and highly precise measurements of protein kinase (and phosphatase) activity in an easy to use, one-step homogeneous format that increases the quality of information and improves throughout, while reducing hands-on time. AssayQuant has validated 167 protein kinase target assays and 27 PhosphoSens® substrates, including both highly-generic and highly-selective sensors. The use of generic sensors allows screening of a large number of recombinant kinases with maximum flexibility, while highly-selective sensors enable quantitative kinase profiling in crude (unfractionated) cell and tissue lysates. The PhosphoSens® platform is ideal for studying inhibitor mechanism of action and potency, enzyme activation or profiling with a wide range of samples, including purified enzymes and crude cell or tissue lysates, to meet the diverse needs of modern drug discovery.

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